Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Prototype recombinant lactic acid bacteria to prevent rotavirus infection

Result description:
The rotaviral antigen VP8* was expressed in probiotic lactobacilli, such as L. casei and L. plantarum, as well as in Lactococcus lactis alanine racemase mutant (alr-). The VP8* fragment was expressed from different promoters and on different cell compartments (cytoplasmatic, cell-wall anchored and secreted). All of them were tested in a mouse model using cholera toxin subunit B (CTB) as an adjuvant. Female Balb/c mice were given intragastrically 1x109c.f.u. of each of recombinant L. lactis and L. casei strains producing VP8* and a significant local immune response was achieved. IgA antibodies against VP8* were detected in stools, intestinal fluids and cultured Peyer's patches at higher levels in mice given recombinant bacteria secreting VP8*. Protection against rotavirus infection in the adult mouse model was evaluated using the murine EMcN and the simian SA11 rotavirus strains as the challenging viruses. Reduction in viral antigens shed in stools suggests a partial homotypic protection. Antibodies against the VP8* fragment of the rotavirus spike protein VP4 have been shown to block virus entry into the enterocytes and to protect against rotavirus gastroenteritis. Following this rationale, anti-VP8* single chain antibodies (scFv) have been isolated in Escherichia coli and cloned in L. casei, this constitutes a complementary approach with therapeutic implications. Also active anti-NSP4 scFv clones have been isolated, being NSP4 the first viral enterotoxin described. In vivo trails with these synthetic antibodies could be performed in the future.

Potential use and expected benefits:
Rotavirus is the most important viral agent causing gastroenteritis worldwide and kills 440,000 children each year in developing countries. Therefore, the development of a cost-effective vaccine vehicle, easy to administrate and resistant to harsh manipulation/transport conditions, together with appropriate nutrition and vaccination programmes could notably help to paliate the effects of these infections. The development of lactic acid bacteria vehicles to deliver rotaviral antigens could fit this profile. Furthermore, probiotic strains of Lactobacillus casei with demonstrated anti-rotavirus activity, as well as recombinant strains expressing anti-rotaviral scFv, could reinforce the protecting effect of the recombinant vaccine.

In addition to scientific publications, the existence of this project has been communicated through two personal interviews with local journalists, submission of the project profile to various databases and the corporative web pages of the University of Valencia and Instituto de Agroquímica y Tecnología de Alimentos (CSIC). Additionally, the project structure and our results have been communicated to the local Food Products Fair Interaliment (2001).

Key innovative features of the results:
The results obtained have several relevant innovative aspects related to the location and expression of heterologous proteins in lactic acid bacteria and to the immune response in mice. Difficulties were found to achieve a significant expression of rotavirus capsid protein in lactic acid bacteria, followed by little success in the first immunisation assays in mice. It was evidenced that previously published successful models might not be "the rule". However, this forced the team to test a number of expression vectors, bacterial hosts and inoculation protocols, adjuvants and even mice strains, until the system was put to work. Also, this work reported the isolation and cloning of anti-viral scFv in lactic acid bacteria.

Current status and use of results:
At present, the last protection experiments are performed in mice, however, in vivo tests with the cloned antibodies or other likely therapeutic molecules are yet to be done. After publication of the results, it would be the time to seek further financing sources and a potential industrial partner interested in the development of further research leading to particular applications in humans and possibly in farm animals.

Our research work has proved that an oral immunisation method in mice can be developed for rotaviral antigens that initially offered serious difficulties. Despite the fact that other antigens secreted by lactic acid bacteria have been proven to elicit systemic and mucosal immune response, the underlying mechanisms are totally unknown. Particularly, we would seek collaboration with immunologists to determine the causes of the null mucosal immune response to the VP8* peptide, either purified or expressed in lactic acid bacteria. Since differences in the behaviour and response in different animal species could be expected, further research and networking would also allow to adapt and test the system in farm or experimental animals other than mice, before considering human trails. Existing "clean" recombination methods can be implemented to yield antibiotic-free and other environmentally safe strains (i.e., self-contained). The methods developed and knowledge obtained will facilitate the development of live innocuous bacterial vectors to deliver other antigens (as mucosal vaccines) or therapeutic molecules for many other applications. They seem to be particularly suited for the prevention of gastrointestinal disorders or mucosal infections.

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